In Vitro Screening Method for Characterization of Macrophage Activation Responses

Macrophage activation refers to the enhanced functionality of macrophages in response to endogenous or exogenous stimuli. Due to the existence of limitless stimuli and a multitude of receptors on macrophage surfaces, the nature of activation (or acquired functioning) can be specific to the encountering stimulus. This article describes a macrophage-activation screening platform in a 96-well format. The methodology involves the generation of bone marrow-derived macrophages, their activation into two extreme activation states, and screening of activated macrophages for expression of bonafide protein biomarkers. A high-throughput and stringent assay to determine macrophage activation markers developed in this article can be adapted for biomarker determination in pathological conditions and toxicant/drug safety screening.

Identification of macrophage activation patterns can be performed in purified macrophages from healthy and diseased tissues using gene expression profiling [15,16], western blotting, immuno-cytochemical staining, and flow cytometry [17,18]. Although employed routinely, these approaches generally pose challenges, including the selection of effective reagents (antibodies), tissue/cell processing, and lack of positive controls. In our attempt to develop a robust, reliable, and consistent assay, after screening various antibody specificities using protein electrophoresis/western assays, we employed an in-cell western technology to develop a macrophage activation screening platform. In-cell western offers many advantages compared to traditional assays such as immuno-cytochemistry and western blotting. It provides higher sensitivity and more meaningful protein expression analyses in whole cells. This approach allows for enhanced quantitative analyses in a high throughput manner in plated whole cells. Importantly, this approach allows reduction in the number of animals used in research. Although developed using BMDM cell line, the assay can be adapted to various primary macrophage (Bronchoalveolar/peritoneal macrophages) and immortalized macrophage cell lines. Table 1 summarizes the reagents and equipment utilized in this study. An overview of experimental procedure describing generation of BMDMs is shown in Figure 1.

Generation of Macrophage-Colony Stimulating Factor (M-CSF) Containing L929 Medium
An overview of experimental procedure describing generation of BMDMs is shown in Figure 1.

Collection of Bone Marrow Cells
(1) Euthanize six-week-old male mice on C57BL/6J background by CO2 inhalation and

Collection of Bone Marrow Cells
(1) Euthanize six-week-old male mice on C57BL/6J background by CO 2 inhalation and cervical dislocation. Disinfect euthanized mice with 70% ethanol and betadine (this step can be performed on bench-top). (2) Using sterile surgical instruments, isolate femur and tibial bones, and transfer to 100 mm cell culture dish containing 5 mL of RPMI 1640 complete media (10% FBS, and 1% pen/strep).
(3) Transfer cell culture dish containing bones to cell culture hood for further processing. Using new set of surgical instruments, cut bones from both ends to expose the marrow cavity. Flush the bone marrow into new cell culture dish with 10 mL syringe (containing RPMI 1640 complete media) fitted with 22G needle. (4) Dissociate bone marrow with repeated passing through 25G needle. Strain the cell suspension through 70 µm cell strainer in to 50 mL sterilized conical tube.
(5) Spin the cell suspension at 400× g for 5 min. Resuspend the cell pellet in 20 mL of L929-conditioned macrophage medium and plate in T25 cell culture flask for overnight incubation. Next morning (~12 h wait), replate non-adherent cells from T25 flask into 100 mm petri dishes (20 dishes per mouse) for differentiation of bone marrow cells to macrophages. (6) Add 3 mL fresh L929-conditioned macrophage medium every day. Switch L929conditioned macrophage medium at 3-day intervals (7 mL per dish). Collect adherent cells (mature macrophages) on 10th day of culture and proceed to in vitro macrophage activation step.

BMDM Macrophage Activation
A step-by-step protocol for generation of activated macrophages is shown in Figure 2.
( cells (mature macrophages) on 10th day of culture and proceed to in vitro macrophage activation step.

BMDM Macrophage Activation
A step-by-step protocol for generation of activated macrophages is shown in Figure  2.
(1) Seed BMDMs in designated wells (seeding density: 40,000 per well in 200 μL volume) of 96-well cell culture plate. Incubate the plate at 37 °C for 1 h. (2) To prevent the likely influence of serum constituents on the macrophage responsiveness at baseline, replace the media with serum-free RPMI 1640 complete medium, and incubate the plate for overnight serum starvation. Of note, in case of possible loss of viability of cells due to serum-free environment, RPMI 1640 complete medium with 1-2% serum could be used. (3) Next day, label wells for M1, M2, and M0 (non-activated). Replace media (24 wells/treatment) with fresh serum-free RPMI 1640 complete medium containing IFNγ (215 U/mL) + LPS (10 ng/mL; added 8 h after the start of IFN-γ treatment) for M1 activation or IL-4 (20 U/mL) for M2 activation. Wells assigned to M0 group will receive fresh serum-free RPMI1640 complete medium with no additives [8].

Screening for Macrophage Activation Markers
A detailed overview of macrophage activation screening is shown in Figure 3.

Screening for Macrophage Activation Markers
A detailed overview of macrophage activation screening is shown in Figure 3.
(1) Wash once with 1× PBS buffer and fix with 100 µL of 10% neutral buffered formalin (Fisher Scientific) for 20 min, at room temperature. Use of multichannel pipette is highly recommended. To avoid dislodgment of cells, it is important to add buffers/solutions alongside the walls. (1) Wash once with 1× PBS buffer and fix with 100 μL of 10% neutral buffered formalin (Fisher Scientific) for 20 min, at room temperature. Use of multichannel pipette is highly recommended. To avoid dislodgment of cells, it is important to add buffers/solutions alongside the walls.

Results
First, we tested the specificity of antibodies for bonafide M1 and M2 markers by performing western blots (Figure 4). The protein expression of INOS, a bonafide marker to test M1 activation, was enhanced in classically activated macrophages, whereas the protein expression of FIZZ1, a bonafide marker to test M2 activation, was enhanced in alternatively activated macrophages.    INOS (M1 marker) and FIZZ1 (M2 marker) expression status was interrogated in cell western approach ( Figure 5). Similar to the results obtained with western blots ure 4), INOS was enhanced in classically activated macrophages while FIZZ1 wa hanced in alternatively activated macrophages in in cell western assays. Antibody specificity for other activation markers (ARG1, COX1, MM ALOX12/15, and YM1/2) was confirmed using western blot (data not shown). Finall tivated macrophages were interrogated for the expression levels of selective activ markers ( Figure 6). Antibody specificity for other activation markers (ARG1, COX1, MMP12, ALOX12/15, and YM1/2) was confirmed using western blot (data not shown). Finally, activated macrophages were interrogated for the expression levels of selective activation markers ( Figure 6).

Discussion
In this article, we report a well-standardized and highly adaptive in vitro method to assess macrophage activation patterns based on the expression of activation-specific protein markers. Although the current study is focused on BMDM activation responses to M1 and M2 treatments, the method can be easily modified for any other macrophages, including other primary macrophages and macrophage cell-lines. In addition, the method offers multiple advantages in research. First, the 96-well format allows simultaneous screening of large number of test samples. Additionally, few wells of 96-well plates can be seeded with known activated and naïve macrophages that can serve as positive and negative controls during analyses of unknown samples. Second, the method can be adapted for high

Discussion
In this article, we report a well-standardized and highly adaptive in vitro method to assess macrophage activation patterns based on the expression of activation-specific protein markers. Although the current study is focused on BMDM activation responses to M1 and M2 treatments, the method can be easily modified for any other macrophages, including other primary macrophages and macrophage cell-lines. In addition, the method offers multiple advantages in research. First, the 96-well format allows simultaneous screening of large number of test samples. Additionally, few wells of 96-well plates can be seeded with known activated and naïve macrophages that can serve as positive and negative controls during analyses of unknown samples. Second, the method can be adapted for high throughput screening of drugs/toxicants for safety assessment. Third, the method can be adapted for phenotypic investigation of macrophage populations from various murine models of diseases.
One caveat of this method is the inability to simultaneous label cells for more than one marker. This limitation is commonly encountered in most of the current techniques except flow cytometry. However, the in vitro method described in this study presents a robust tool as a first line of investigation on macrophage responses in toxicant/drug screening as well as disease investigation. While this protocol was established using murine BMDMs, it can be used for other types of macrophages, including human monocytes derived from peripheral blood mononuclear cells and macrophages derived from other species. However, differentiation of mononuclear cells into macrophages would require species-specific protocols.
Inducible nitric oxide synthase (INOS) is the signature M1-activation marker that is characteristic of classical activation response [19]. As expected, INOS expression was only observed in classically activated macrophages. For M2 activation, we specifically focused on M2a subset that is induced by IL-4. Our method can be adapted to macrophages stimulated with TFGβ and IL-10 (M2c). For M2-specific signatures, we used found in inflammatory zone 1 (FIZZ1) and chitinase 3 like 4 (CHI3L/4) markers [12,20]. FIZZ1, a well-standardized M2 marker, was only seen in M2 macrophages. Although baseline expression of CHI3L3/4 (also known as YM1/2) was observed in M0 as well as M1 macrophages, only M2 macrophages showed significantly upregulated expression. The assessment of the expression levels of these three markers would identify M1 or M2-activated macrophages.
In summary, the assay developed in this article is highly adaptable, high-throughput, and informative and can be utilized in toxicological screening as well as disease phenotyping studies.